Mass spectrometers are often coupled with chromatography systems in order to identify and characterize eluting species from a test sample. In such a coupled system, the eluting solvent is ionized and a series of mass spectrograms are obtained of the eluting solvent at specified time intervals, ranging from, for example, 0.01–10 seconds, for subsequent data analysis. As the test sample may contain many species or compounds, it is often desirable to be able to automatically determine or identify species or compounds of interest as they elute and perform MS/MS analysis to characterize them. However, identifying species of interest in complex mixtures in real time can be a challenging task.
A variety of automation tools and data acquisition & analysis software associated with mass spectrometers have been developed to achieve this goal. A well known automation tool is the Information Dependant Acquisition™ (IDA™) system marketed by MDS Sciex Inc. and Applera Corporation. During the data acquisition process this tool identifies a mass peak in a mass spectrogram so as to select a precursor ion. The tools thus directs one or more subsequent stages of mass spectrometry (MS/MS or MS/MS/MS) in which the chosen precursor ion is fragmented. The resulting MS/MS (or higher) spectrum is a composite of all the fragmentation processes that are energetically allowed: precursor ion to fragment ion and fragment ions to other fragment ions. This spectral richness and/or the dissociation pathways elucidated by subsequent MS stages can be quite useful for identifying compounds when searching through spectral databases or MS/MS libraries or providing structural information used in characterizing compounds.
Vendors of other mass spectrometer systems provide similar real time data dependent switching functions. For example, Thermo Finnigan LLC of San Jose, Calif., markets the Data Dependent Experiment™ (DDE) tool and Waters Corporation (Micromass™) markets the Data Directed Analysis (DDA) tool.
The above-mentioned real time data dependent switching functions provide good results in applications such as in-vitro sample analysis or single protein digest analysis where it is possible to a detect a mass peak of interest fairly easy. However, when dealing with a more complex sample set such as a biological fluid, (e.g., urine or plasma extracts) or mixtures of digested proteins (e.g., digested cell lysate), there may be many other major components or species eluting at the same time that will often “shadow” or hide the signal of the analyte or species of interest, which may have weaker signal intensities, thus making it impossible to effectively select the (ionized) species of real interest. In essence, it is often difficult to automatically detect species eluting at a low level of concentration.
In the IDA™ tool, the selection of the mass peak ‘chosen’ by the system for MS/MS can be improved by relying on a use and inclusion list or by using more specific survey scans such as neutral loss and precursor scans, as known in the art per se. However, these approaches preclude some knowledge of the sample to be analyzed, which is not always the case. Alternatively, a dynamic exclusion process can be activated wherein, once an ion has been selected for dissociation, that ion is ignored over the next few scans such that the ion having the next most significant intensity peak is selected for dissociation. However, this does not solve the problem of weakly concentrated species that elute simultaneously with a number of other major components.
It will be appreciated that the proper selection of precursor ions is an important step in species identification. The proper selection of ions will also ensure that a useful yet minimal amount of information is collected in data dependent acquisition techniques, which can assist in speeding up and simplifying species identification and characterization.